Probability of piRNA 5′-to-5′ distances on the same genomic strand for a representative set of 35 animal species. Distance probability analysis was done for ≥24-nt sequencing reads without taking into account abundance. Red: data smoothed using non-
parametric regression (LOWESS). The insets show autocorrelation analysis of the smoothed data. The periodicity interval (i.e., estimated pre-piRNA length) was identified either as the peak of the autocorrelation function or the length closest to the zero data point of the derivative of the autocorrelation function. All data are from wild-type
animals, except for Mus musculus (Pnldc1−/−). Figure S2, related to Figures 3 and 4
(A) Probability of 3′-to-5′ distances on the same genomic strand for ≥24-nt small RNAs in BmN4 cells. Either Trimmer/PNLDC1 (piRNA trimming enzyme) or Renilla luciferase (control) were depleted using RNAi (Izumi et al., 2016).
(B) Probability of distances from the 3′ ends of ≥24-nt small RNAs in primary spermatocytes of wild-type or Pnldc1−/− mice to the 5′ ends of ≥150 nt long
5′ monophosphorylated RNAs in primary spermatocytes of wild-type mice. Data are from a single representative biological sample.
(C, D) Relative abundance of MILI (C) and MIWI (D) in male germ cells wild-type and
Pnldc1–/– mice assessed by Western blotting. Spg, spermatogonia; SpI, primary spermatocytes; SpII, secondary spermatocytes; RS, round spermatids. Each lane contains lysate from ~11,000 cells. Data are from three biological samples.
(E) Probability of 5′-to-5′ distances on the same genomic strand for ≥24-nt small RNAs in spermatogonia and primary spermatocytes of wild-type mice. Data are from two biological samples.
(F) Strategy to analyze 3′ ends of pre-piRNAs deriving from the same pre-pre-piRNA. First, pre-piRNAs with common 5′, 25-nt prefix are grouped and the read abundance is used to identify the most frequent 3′ end in each group. Second, corresponding MILI- and MIWI-bound pre-piRNA groups are paired and the distance is calculated between the most frequent 3′ end in MILI group and the most frequent 3′ end in MIWI group. If this distance is 0, the paired group falls into cohort 0; if the most frequent 3′ end in MILI group is 1 nt upstream of the most frequent 3′ end in MIWI group, the paired groups falls into cohort 1, etc.
Figure S3, related to Figure 5
(A) Nucleotide bias for the 5′ section of MILI- or MIWI-bound piRNAs in wild-type and the same for pre-piRNAs in Pnldc1−/− primary spermatocytes.
(B) Probability of a single uridine surrounded by non-uridine stretches (VVVVUVVVV), and probabilities of lengths of non-uridine nucleotide stretches between two nearest uridines (UU, UVU, UVVU, etc.) in pachytene piRNA loci estimated by random sampling of the data from pachytene piRNA cluster transcripts (grey bars). Mean ± standard deviation are presented for the randomly sampled data based on 1,000 iterations. Data for pre-piRNA cohorts (pink bars) are for a single biological sample from Figure 4C. (C) The first prediction of the model for pre-piRNA production in which MILI and MIWI proteins direct endonucleolytic cleavage by binding 5′ ends of pre-pre-piRNAs: protein footprints limit the availability of uridines for the pre-pre-piRNA cleaving endonuclease (vertical arrows). Because MILI footprint is smaller than that of MIWI, the endonuclease will have access to more upstream uridines if pre-pre-piRNA is bound by MILI compared
to MIWI. MILI footprint will still place a 5′ limit on the upstream shift in the uridine availability windows for MILI and MIWI.
(D) Test of the first prediction (Figure S3C) of the model for pre-piRNA production. Upper panel, strategy to create sets of simulated pre-piRNAs. Uridines randomly sampled in pachytene piRNA clusters were used as 5′ ends of simulated pre-piRNAs. The 3′ ends of the simulated pre-piRNAs were set immediately before the first uridine occurring >31 nt (simulated MILI footprint) or >34 nt (simulated MIWI footprint)
downstream of the simulated pre-piRNA 5′ end. Lower panel, comparison of cohort sizes calculated from simulated and biological data. Biological data are from Figure 4C. Mean ± standard deviation are presented for the simulated data based on 1,000
iterations.
(E) The second prediction of the model for pre-piRNA production in which MILI and MIWI proteins direct endonucleolytic cleavage by binding 5′ ends of pre-pre-piRNAs: MILI-bound pre-piRNAs present in cohorts ≥4 are paired with atypically long MIWI- bound pre-piRNAs because of the limitation on the minimal length of a MILI-bound pre- piRNA imposed by the MILI footprint.
(F) Test of the second prediction (Figure S3E) of the model for pre-piRNA production. Length of the corresponding paired MILI- and MIWI-bound pre-piRNA groups in
Pnldc1−/− primary spermatocytes in cohorts 0–9 (Figure 5). Data are from a single representative biological sample. Whiskers correspond to minimum and maximum values. Nucleotide bias of the genomic neighborhood around the 3′ ends of paired MILI- and MIWI-bound pre-piRNA in each cohort is shown.
Figure S4
(A) Absolute abundance of miRNAs, small RNAs (24−33-nt for wild-type; 24−45-nt for
Pnldc1−/− mice. Data are presented as the mean ± standard deviation from two biological replicates for small RNAs and from three biological replicates for MIWI proteins. Median cell volume was used to calculate cellular concentration of 24−45-nt long RNAs and PIWI proteins.
(B, C) Abundance of MILI (B) and MIWI (C) in ~11,000 wild-type germ cells relative to the standards of SNAP-tagged PIWI proteins assessed by Western blotting. SpI, primary spermatocytes; SpII, secondary spermatocytes; RS, round spermatids. Data are from three biological samples.
(D) Correlation between simulated and biological piRNA length distributions. Simulated profiles were created by combining MILI- and MIWI-bound piRNA length distributions at different ratios. Data are from a single representative biological sample.
Figure S5, related to Figure 6
(A) Probability of distances from the 3′ ends of Piwi- or Aub-bound piRNAs to the 5′ ends of Piwi- or Aub-bound piRNAs on the same genomic strand in wild-type D.
melanogaster ovary. Data are for all genome mapping piRNAs from a single
representative biological sample.
(B) Probability of distances from the 5′ ends of Piwi-bound piRNAs to the 5′ ends of
Aub-bound piRNAs on the same genomic strand, and from the 3′ ends of Piwi-bound
piRNAs to the 3′ ends of Aub-bound piRNAs on the same genomic strand in wild-type
D. melanogaster ovary. Numbers indicate the total probability of 5′ or 3′ ends of Aub- bound piRNAs residing before, after or coinciding with the 5′ or 3′ ends of the Piwi- bound piRNAs. Data are for all genome mapping piRNAs from a single representative biological sample.
(C) Distance between the most frequent 3′ end of the Piwi-bound piRNA group and the most frequent 3′ end of the corresponding paired Aub-bound piRNA group in wild-type
D. melanogaster ovary. Data are for all unambiguously mapping piRNAs from a single
representative biological sample.
(D) Probability of distances from the 3′ ends of Piwi- or Aub-bound pre-piRNAs to the 5′ ends of Piwi- or Aub-bound pre-piRNAs on the same genomic strand for papi−/− and the same for nibbler−/− D. melanogaster ovary. Data are for all genome mapping pre- piRNAs from a single representative biological sample.
Figure S6, related to Figure 7
(A) Probability of distance from the 5′ ends of MILI- or MIWI-bound piRNAs to the 5′ ends of long 5′ monophosphorylated RNAs in wild-type primary spermatocytes. Data are for piRNAs deriving from pachytene piRNA loci from a single representative
biological sample.
(B) Probability of distances between the 5′ ends of long RNAs (putative pre-pre-piRNAs or control) and the 5′ ends of MILI- or MIWI-bound piRNAs on opposite genomic
strands. Only nucleotides 2 to 10 of guide piRNAs (g2–g10) were required to be complementary to the target long RNAs. Data are for MILI- or MIWI-bound piRNAs derived from pachytene piRNA loci for a single biological sample and randomly resampled long RNAs derived from pachytene piRNA loci from the same biological sample. Mean ± standard deviation of 1,000 resampling iterations are presented. (C) Percent of piRNAs explaining the 5′ ends of either putative pre-pre-piRNAs or the control RNAs. Only nucleotides 2 to 10 of guide piRNAs (g2–g10) were required to be complementary to the target long RNAs. Data are for MILI- or MIWI-bound piRNAs derived from pachytene piRNA loci for a single biological sample and randomly resampled long RNAs derived from pachytene piRNA loci from the same biological sample. Data are from 1,000 resampling iterations. Whiskers correspond to minimum
and maximum values. Wilcoxon rank-sum test was used to assess statistical significance.
(D) Pre-piRNAs sorted by abundance and divided into 10 equally sized bins. At left, percent of total pre-piRNA abundance and the range of read abundance in each bin is shown. In the center, percent of the initial uridine among putative pre-pre-piRNAs sharing their 5′ ends with pre-piRNA bins. Percent of the initial uridine for all putative pre-pre-piRNAs is shown as the vertical dashed line. At right, ratio of the total pre- piRNA abundance to the total pre-pre-piRNA abundance in each bin.
STAR Methods Mice
C57BL/6J mice (IMSR Cat# JAX:000664, RRID:IMSR_JAX:000664) were maintained and used according to the guidelines of the Institutional Animal Care and Use
Committee of the University of Massachusetts Medical School. Mili−/− mice (IMSR Cat# RBRC09475, RRID:IMSR_RBRC09475) were a gift from Dr. Shinichiro Chuma (Kyoto University, Japan).
To create Pnldc1−/− mutants, three sgRNAs targeting sequences in exon 1, exon
2, and intron 2 of Pnldc1 (5′-GUC CCA GGG CGC GCC GGA UGC GG)-3′, 5′-UGU
CUC GGC CCC AAC AGA UCA GG)-3′, and 5′-UAA CUA AGG AGA CAC CGG UGA
GG-3′, underlining denotes the PAM) were transcribed using T7 RNA Polymerase and
purified by electrophoresis through a 10% polyacrylamide gel. Superovulated female C57BL/6J mice (7–8 weeks old) were mated to C57BL/6J stud males, and fertilized embryos were collected from oviducts. A mix of Cas9 mRNA (50 ng/µl, TriLink
Biotechnologies, L-7206) and the three sgRNAs (each 20 ng/µl) were injected into the cytoplasm or pronucleus of fertilized eggs in M2 medium (Sigma, M7167). The injected zygotes were cultured in KSOM with amino acids at 37°C under 5% CO2 until the
blastocyst stage (3.5 days). Thereafter, 15–25 blastocysts were transferred into the uterus of pseudopregnant ICR females at 2.5 dpc.
For genotyping, genomic DNA extracted from tail tissues was analyzed by PCR
using primers 5′-TTC CCA GCA TGA GAA GAT CA-3′ and 5′-CCA CTC AGA TGG CAA
GTC AA-3′. PCR products were Sanger-sequenced using the sequencing primer 5′-
TGA CAC GTG CAC GAG CTT TA-3′. The male sterility phenotype reported previously
(Ding et al., 2017; Zhang et al., 2017) was confirmed by the presence of epididymal sperm, testis weight, and histological assessment of the testes.
Isolation of Mouse Germ Cells by FACS
Testes were isolated, decapsulated, and rotated in 1× Gey′s Balanced Salt Solution (GBSS, Sigma, G9779) containing 0.4 mg/ml collagenase type 4 (Worthington LS004188) at 150 rpm for 15 min at 33°C. Seminiferous tubules were then washed twice with 1× GBSS and rotated in 1× GBSS with 0.5 mg/ml Trypsin and 1 µg/ml
DNase I at 150 rpm for 15 min at 33°C. After incubation, the tubules were homogenized by pipetting with a Pasteur pipette for 3 min on ice, and fetal bovine serum (FBS; 7.5% f.c., v/v) was added to inactivate trypsin. The cell suspension was then strained through a pre-wetted 70 µm cell strainer and pelleted at 300× g for 10 min. The supernatant was removed, cells were resuspended in 1× GBSS containing 5% (v/v) FBS, 1 µg/ml DNase I, and 5 μgl/ml Hoechst 33342 (Thermo Fisher, 62249) and rotated at 150 rpm for 45 min at 33ºC. Propidium iodide (0.2 μg/ml, f.c.; Thermo Fisher, P3566) was added, and cells strained through a single pre-wetted 40 µm cell strainer. Cell sorting at the UMass Medical School FACS Core was performed as described previously (Bastos et al., 2005; Cole et al., 2014). Cell viability was assessed using live phase contrast
microscopy. Purity check of sorted fractions was performed by immunostaining aliquots of cells.
For immunostaining, cells were incubated for 20 min in 25 mM sucrose and then fixed on a slide with 1% (w/v) paraformaldehyde solution containing 0.15% (v/v)
Triton X-100 for 2 h at room temperature in a humidifying chamber. Slides were then sequentially washed for 10 min in (1) 1× PBS containing 0.4% (v/v) Photo-Flo 200 (Kodak, 1464510), (2) 1× PBS containing 0.1% (v/v) Triton X-100, (3) 1× PBS
containing 0.3% (w/v) BSA, 1% (v/v) donkey serum (Sigma, D9663), and 0.05% (v/v) Triton X-100. After washing, slides were incubated with primary antibodies in 1× PBS containing 3% (w/v) BSA, 10% (v/v) donkey serum, and 0.5% (v/v) Triton X-100 in a humidifying chamber overnight at room temperature. Primary antibodies used in this
study were rabbit polyclonal anti-SYCP3 (Abcam Cat# ab15093, RRID:AB_301639, 1:1000 dilution), and mouse monoclonal anti-γH2AX (Millipore Cat# 05-636,
RRID:AB_309864, 1:1000 dilution). Slides were washed again as described above and incubated with secondary donkey anti-mouse IgG (H+L) Alexa Fluor 594 (Thermo Fisher Scientific Cat# A-21203, RRID:AB_2535789, 1:2000 dilution) or donkey anti- rabbit IgG (H+L) Alexa Fluor 488 (Thermo Fisher Scientific Cat# A-21206,
RRID:AB_2535792, 1:2000 dilution) antibodies for 1 h in a humidifying chamber at room temperature. After incubation, slides were washed three times for 10 min in 1× PBS containing 0.4% (v/v) Photo-Flo 200 and once for 10 min in 0.4% (v/v) Photo-Flo 200. Finally, slides were dried, mounted with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher, P36931), and covered with a cover slip.
SNAP-tagged MIWI Protein Standard
SNAP-tagged Mus musculus PIWIL1 (SNAP-MIWI) was produced in HEK293T cells from a lentiviral-transduced transgene (lentivirus backbone was a gift from Greiner Lab). Cells, washed twice with PBS, were homogenized in lysis buffer (30 mM HEPES-KOH pH 7.5, 100 mM potassium acetate, 3.5 mM magnesium acetate, 1 mM dithiothreitol, 20% (w/v) glycerol, 0.1% (v/v) Triton X-100, 1 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride, 0.3 µM Aprotinin, 40 µM Bestatin, 10 µM E-64, 10 µM
Leupeptin). Lysate was centrifuged at 20,000 × g for 30 min, and the supernatant
aliquoted and stored at −80°C. SNAP-MIWI was labeled with SNAP substrate SNAP-
Surface 549 (NEB, S9112) and resolved by electrophoresis through a 4–20% gradient SDS-polyacrylamide gel (Bio-Rad Laboratories, 5671085). The concentration of full- length SNAP-MIWI was determined by comparison to a standard curve of purified, SNAP-Surface 549 labeled SNAP protein (Typhoon FLA 7000; GE Lifesciences). The concentration of purified SNAP protein (gift from Moore Lab) was determined before labeling by BCA assay and by measuring its absorbance at 280 nm (ε = 20970 M-1 cm-1
in water assuming all Cys residues are reduced). SNAP-MIWI of known concentration was then used to estimate the number of MIWI molecules present in FACS-purified germ cells from mouse testes by quantitative immunoblotting using anti-MIWI antibody. Western Blotting
Cells were homogenized in lysis buffer (20 mM Tris-HCl pH 7.5, 2.5 mM MgCl2, 200 mM
NaCl, 0.05% (v/v) NP-40, 0.1 mM EDTA, 1 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride, 0.3 µM Aprotinin, 40 µM Bestatin, 10 µM E-64, 10 µM Leupeptin) and centrifuged at 20,000 × g for 20 min at 4°C. The supernatant was moved to a new tube, an equal volume of loading dye (120 mM Tris-Cl, pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 2.5% (v/v) 2-Mercaptoethanol, 0.2% (w/v) bromophenol blue) was added, the sample incubated at 90°C for 5 min and resolved through a 4–20% gradient
polyacrylamide/SDS gel electrophoresis (Bio-Rad Laboratories, 5671085). After
electrophoresis, proteins were transferred to PVDF membrane (Millipore, IPVH00010), the membrane blocked in Blocking Buffer (Rockland Immunochemicals, MB-070) at room temperature for 2 h and then incubated overnight at 4°C in Blocking Buffer containing primary antibody (anti-PIWIL2/MILI, Abcam Cat# ab36764,
RRID:AB_777284, 1:1000 dilution; anti-PIWIL1/MIWI, Abcam Cat# ab12337,
RRID:AB_470241, 1:1000 dilution). Next, the membrane was washed three times with Blocking Buffer at room temperature for 30 min and incubated for 2 h at room
temperature with donkey anti-rabbit IRDye 680RD secondary antibody (LI-COR
Biosciences Cat# 926-68073, RRID:AB_1095444, diluted 1:20,000) in Blocking Buffer. Then the membrane was washed three times with Blocking Buffer at room temperature for 30 min and the signal was detected using Odyssey Infrared Imaging System. Data was obtained for two independent biological replicates.
Small RNA Immunoprecipitation
Mouse total testis or sorted germ cells were homogenized with lysis buffer (20 mM Tris-
HCl, pH 7.5, 2.5 mM MgCl2, 200 mM NaCl, 0.05% (v/v) NP-40, 0.1 mM EDTA, 1 mM 4-
(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride, 0.3 µM Aprotinin, 40 µM
Bestatin, 10 µM E-64, 10 µM Leupeptin) and then centrifuged at 20,000 × g for 20 min at 4°C, retaining the supernatant. Anti-MIWI (Wako, Cat# 017-23451,
RRID:AB_2721829, ~5 µg; Nishibu, 2012) or anti-MILI (Abcam Cat# ab36764,
RRID:AB_777284, ~5 µg) antibodies were incubated with rotation with 30 µl of Protein G Dynabeads (Thermo Fisher, 10003D) in 1× PBS containing 0.02% (v/v) Tween 20 (PBST) at 4°C for 1 h. The bead-antibody complex was washed with PBST. Freshly prepared testis or cell lysate were added to the bead-antibody complex and incubated with rotation at 4°C overnight. The next day, the beads were washed once with lysis buffer and three times with 0.1 M Trisodium Citrate. After washing, RNA was purified with Trizol reagent (Thermo Fisher, 15596026) and used for small RNA library
preparation. Each experiment was conducted for two independent biological replicates. The specificity of the commercial anti-MILI antibody was confirmed by
immunoprecipitation from lysate of Mili−/− whole testis (data not shown).
Small RNA-seq Library Preparation and Analysis of Small RNA Data Sets
Total RNA from sorted mouse germ cells was extracted using mirVana miRNA isolation kit (Thermo Fisher, AM1560). Small RNA libraries were constructed as described (Han et al., 2015) with several modifications. Briefly, before library preparation, a set of 18 synthetic RNA oligonucleotides was added to each RNA sample to enable absolute quantification of small RNAs (Table S2A). To reduce ligation bias, a 3′ adaptor with
three random nucleotides at its 5′ end was used (5′-rApp NNN TGG AAT TCT CGG
GTG CCA AGG /ddC/-3′). After 3′ adaptor ligation, RNA was purified by 15% urea polyacrylamide gel electrophoresis (PAGE), selecting for 15–55 nt small RNAs (i.e., 40–
80 nt with 3′ adaptor). Small RNA-seq libraries for two independent biological replicates were sequenced using a NextSeq 500 (Illumina) to obtain 75 nt, single-end reads.
The sequence of the 3′ adapter, including the three random nucleotides, was removed from raw reads, which were further filtered by requiring their Phred quality score to be ≥20 for all nucleotides. Sequences of synthetic spike-in oligonucleotides were identified allowing no mismatches (Table S2A). Absolute quantity of small RNAs per library was calculated based on the read abundance of 2′-O-methylated synthetic spike-in oligonucleotides (Table S2B). Reads not fully matching the genome were analyzed using the Tailor pipeline (Chou et al., 2015) to account for non-templated tailing of small RNAs.
Drosophila melanogaster Piwi- and Aub-bound small RNA data sets used in this
study are listed in Table S3A. Distances between small RNAs were calculated using all possible alignments of either in all genome-matching reads (mouse and fly data sets) or matching only to annotated pachytene piRNA loci (mouse data sets only; Li et al.,
2013), taking into account the number of times a small RNA sequence occurs in the library divided by the number of locations where this small RNA maps in the genome (i.e., multi-mapping reads were apportioned). Z0 scores for phasing were calculated as
described (Han et al., 2015) using distances from –10 to –1 and from 1 to 50 as background. Sequence motif charts for genomic neighborhoods around 5′ and 3′ ends of small RNAs were generated with motifStack (Ou et al., 2018) using alignments in pre- pachytene or pachytene piRNA loci only (Li et al., 2013) and apportioning reads.
Grouping of piRNAs or pre-piRNAs (≥1 ppm) with the same 5′, 23-nt or 25-nt prefix was done for all unambiguously mapping piRNAs (mouse and fly data sets) or for those mapping to the pachytene piRNA loci only (mouse data sets only; Li et al., 2013). The most frequent 3′ end in each group was identified based on the number of times 3′ ends are found in the library. Pairing MILI- and MIWI-bound small RNA groups was
done based on their 5′, 25-nt prefix. Pairing Aub- and Piwi-bound small RNA groups was done based on their 5′, 23-nt prefix.
Distance probability analyses of previously published datasets from 34 animal species (Table S3B; Grimson et al., 2008; Friedländer et al., 2009; Kamminga et al., 2010; Song et al., 2012; Li et al., 2013; Juliano et al., 2014; Hirano et al., 2014; Moran et al., 2014; Xu et al., 2014; Han et al., 2015; Williams et al., 2015; Sarkies et al., 2015; Roovers et al., 2015; Rosenkranz et al., 2015; Toombs et al., 2017; Lewis et al., 2017; Fu et al., 2018) were done for ≥24-nt sequencing reads without taking into account their abundance. Smoothing of the data points [20-200] with non-parametric regression (LOWESS) was conducted in R without robustifying iterations and the span set at 0.1. Ping-pong Z10 score was calculated as described (Zhang et al., 2011) for ≥24-nt
sequencing reads without taking into account their abundance using distances from 0 to 9 and from 11 to 20 as background.
RNA-seq Library Preparation and Analysis
Total RNA from sorted germ cells was extracted using mirVana miRNA isolation kit (Thermo Fisher, AM1560) and used for library preparation as described previously (Zhang et al., 2012) with several modifications (Fu et al., 2018). Briefly, before library preparation, 1 µl of 1:100 dilution of ERCC spike-in mix 1 (Thermo Fisher, 4456740, LOT00418382) was added to 0.5–1 µg total RNA to enable absolute quantification of mRNA. For ribosomal RNA depletion, RNA was hybridized in 10 µl to a pool of 186